This project will determine the biological function of the many GATC sites found within the bacterial origin (oriC) of DNA replication. Our previous studies have shown that 9 to 14 sites, with 8 totally conserved, are found in each of the 6 sequenced bacterial origins. The 6 bacteria whose origins have been cloned in E. coli all methylate these sites, although many bacteria do not. The adenine residues in these sites are methylated by the E. coli dam methylase, and dam mutants are deficient in mismatch repair. The transformation rate specifically of origin plasmids using the bacterial origin into E. coli dam mutants is 30-fold lower than into wildtype cells. GATC sites thus function in initiation, perhaps by a combination of methylation and mismatch repair events. The Specific Aims of this project include: 1) Determine why origin plasmids transform E. coli dam mutants at a greatly reduced frequency. 2) Isolate and characterize deletion and conditional mutants of the dam locus using transposons, to prove whether the dam methylase is required for cell viability. 3) Use the new oriC-dependent in vitro DNA synthesis system to elucidate the effects of GATC methylation on initiation in origin plasmids. 4) Use directed mutagenesis in vitro of GATC sites to generate deletion and insertion origin mutants specifically in GATC sites, and to generate point mutants in and near the GATC sites. This approach complements the analysis of transformed cells and plasmids from transformed cells in Specific Aim 1. Methylation events are implicated in both DNA repair and in DNA replication in procaryotes, and undermethylation appears to be very important in gene expression in eucaryotes. Loss of regulation of DNA repair, and gene expression, leads to many diseases, including cancer. Thus, this project is directly relevant to the health sciences.